Abstract

BACKGROUND:

Lactobacillus gasseri SBT2055 (LG2055) has been shown to prevent abdominal adiposity, and suppression of lipid absorption is considered a possible mechanism, detail of which, however, are poorly understood. In the present study, we evaluated the effects of LG2055 on fat hydrolysis by determining pancreatic lipase activity and fat emulsion properties in vitro. We also examined whether LG2055 influences fecal fat excretion in humans.

METHODS:

Pancreatic lipase activity was investigated in vitro using an artificially prepared fat emulsion and 4-methylumbelliferyl oleate (4-MUO) as substrates. The concentrations of free fatty acids and 4-methylumbelliferone were quantified. Fat emulsion droplet size was measured using a particle size analyzer. The clinical study was performed as a double-blind, randomized, placebo-controlled trial. Subjects consumed 100 g of fermented milk (FM)/d, either with or without LG2055 supplementation, for seven days. Fecal samples were collected during three-day pre-observational and FM intake periods and fecal fat levels were determined.

RESULTS:

LG2055 dose-dependently suppressed lipase activity in the fat emulsion assay but not in the 4-MUO assay. LG2055 dose-dependently increased fat emulsion droplet size. The effects of LG2055 on lipase activity and fat emulsion properties were increased compared with four other tested strains (Lactobacillus gasseri SBT0317, Lactobacillus gasseri JCM1131T, Lactobacillus. delbrueckii subsp. bulgaricus JCM1002T and Streptococcus thermophilus ATCC19258T). In our clinical study, fecal fat level after FM intake was significantly increased compared with that observed before FM intake in the LG2055-containing active FM group but not the control FM group lacking LG2055.

CONCLUSIONS:

LG2055 increased fat emulsion droplet size, resulting in the suppression of lipase-mediated fat hydrolysis. The influence of LG2055 on the physicochemical properties of fat emulsion provides a mechanism for the probiotic-mediated suppression of lipid absorption and promotion of fecal fat excretion in humans.

TRIAL REGISTRATION:

Suppressive effect of lactic acid bacteria on pancreatic lipase-mediated hydrolysis of triolein in an emulsion. Substrate suspensions (fat emulsion) were incubated with pancreatic lipase (200 U/ml) and lactic acid bacterial cells or orlistat for 30 min at 37°C. After boiling for 2 min, released fatty acids were quantified. The suppressive activity was calculated for fatty acid production, in which the activity in the absence of sample was represented as 100%. (A)Lactobacillus gasseri SBT2055 (LG2055) suppressed the release of fatty acids from fat emulsion in a dose-dependent manner. (B) Comparison of the capacity of various lactic acid bacteria in suppressing fatty acid release. Each of the five bacterial strains (LG2055, Lactobacillus gasseri SBT0317 (LG0317), Lactobacillus gasseri JCM1131T (LG1131T), Lactobacillus delbrueckii subsp. bulgaricus JCM1002T (LB1002T), and Streptococcus thermophilus ATCC19258T (ST19258T)) was added to the lipase reaction solution at 100 μg/ml. Values are means with standard deviations for triplicate experiments. Statistical differences between the strains were analyzed using the Tukey-Kramer post-hoc test and significant differences (P < 0.05) are indicated using different letters.

Effect ofLactobacillus gasseriSBT2055 (LG2055) on pancreatic lipase activity using 4-methylumbelliferyl oleate (4-MUO) as substrate. Various concentrations of LG2055 and orlistat, and 0.1 mM 4-MUO solution were mixed in the well of a microtiter plate, and the lipase solution (50 U/ml) was added. After incubation at 25°C for 30 min, 0.1 M sodium citrate (pH 4.2) was added to terminate the reaction. The suppressive activity was calculated based on the amount of 4-methylumbelliferone, in which the activity in the absence of sample was represented as 100%. Values are means with standard deviations for triplicate experiments.

Effect ofLactobacillus gasseriSBT2055 (LG2055) on fat emulsion droplet sizein vitro. LG2055 or a catechin mixture was added to the fat emulsion preparation. The suspension was incubated at 37°C with constant shaking for 3 h and the size distribution of fat emulsion was measured using a particle size analyzer. No sample (only TES buffer) was added to the negative control and the catechin mixture was used as a positive control. (A) Changes in emulsion droplet size distribution after incubation with 1000 μg/ml of LG2055 or catechin mixture. The distributions are an average of three determinations. (B) Phase-contrast micrographs (scale bar: 50 μm). Black and white arrows indicate the representative fat emulsions and LG2055 cells, respectively.

Increased effects ofLactobacillus gasseriSBT2055 (LG2055) on fat emulsion droplet size. (A) The dose-dependency of LG2055 on the average fat emulsion droplet size. (B) Comparison among lactic acid bacteria at 100 μg/ml. Fat emulsion droplet size was measured in the same manner as described in Figure . Values are means with standard deviations for triplicate experiments. Statistical differences between the strains were analyzed using the Tukey-Kramer post-hoc test and significant differences (P < 0.05) are indicated using different letters.

Effect of intake of FM containingLactobacillus gasseriSBT2055 (LG2055) on fecal fat excretion in humans. (A) Fecal samples were collected during the final three days of the pre-observational period (before) and FM intake period (after), and fecal fat levels were determined. (B) The amount of change in the fecal fat levels in the control and active FM groups. Values were calculated by subtracting fecal fat levels at pre-observational period from those at FM intake period. Values are means with standard deviations. Statistical analysis was performed using paired Student’s t-test (A) and unpaired Student’s t-test (B). An asterisk indicates a significant difference (P < 0.05).